Fas peptides and antibodies for modulating apoptosis

ABSTRACT

Anti-Fas (APO-1, CD95) autoantibodies arm found in human s, which antibodies arm biologically functional. Peptide fragments of Fas recognized by such antibodies, and antibodies specific for such peptides, inhibit or promote apoptosis and cellular proliferation. Assay methods making use of Fas peptides or antibodies enable identification of further agents which modulate apoptosis and/or cellular proliferation.

The present invention relates in various aspects to methods and meansfor modulating apoptosis and/or cellular proliferation, in particularvia stimulation or inhibition of Fas (also known as APO-1 and CD95). Itis based in part on the surprising discovery of anti-Fas autoantibodiesin human sera, which antibodies moreover are biologically functional andinclude both IgG and IgM antibodies. Peptide fragments of Fas andvariants and mimetics thereof may be used in modulating apoptosis fortherapeutic purposes.

Affinity-purified anti-Fas antibodies isolated from the serum of healthyblood donors have been found by the present inventors to be able toinhibit proliferation and to induce apoptosis of Jurkat leukemia Tcells. This effect is inhibited by soluble Fas-Fc chimeric protein.Costimulation of peripheral blood mononuclear cells by human anti-Fasautoantibodies and anti-CD3 monoclonal antibodies induces or inhibitscell proliferation depending on the activation state of the cells.Anti-Pas autoantibodies may thus represent an additional mode ofregulation of Fas-mediated signals in vivo which may be harnessed inaccordance with the present invention.

Fas (also called CD95/APO-1) is a type I cellular receptor protein,belonging to the nerve growth factor/tumor necrosis factor (NGF/TNF)receptor family (Itoh, et a l., 1991; Smith et al., 1994). The receptorhas been shown to transduce an apoptotic signal in various cell typesupon binding of its natural ligand, aas ligand (FasL) (Suda et al.,1993).

Certain anti-Fas monoclonal antibodies have also been shown to triggerFas signaling by crosslinking this receptor in vitro. Anti-Fasantibodies can (i) induce apoptosis of immortalized and chronicallystimulated T and B cells (Yonehara et al., 1989; Trauth et al., 1989;Weis et al., 1995; Mapara et al., 1993; Owen-Schaub et al., 1992; Kiaset al., 1993) and liver cells (Ogasawara et al., 1993); (ii) induceproliferation of peripheral lymphocytes in response to anti-CD3 antibody(Alderson et al., 1993; Alderson et al, 1994); (iii) inhibit apoptosisinduced by either another anti-Fas antibody (Silvestris et al., 1996) orFasL (Alderson et al, 1994). Fas has been suggested to play a major rolein organ-specific destruction during autoimmune conditions andinfectious diseases (Katsikis et al., 1995; Watanabe-Fukunaga et al.,1993; Estaquier, et al., 1995; Galle, et al., 1995; Stassi et al., 1997;Dowling et al., 1996; D'Souza et al., 1996).

Based on the experimental work described below, the present inventionprovides in various aspects methods and means for modulating apoptosisand/or cell proliferation. Inducing apoptosis and/or inhibiting cellproliferation may be used in therapeutic contexts includingproliferative disorders such as tumors, cancer and psoriasis. Inhibitingapoptosis may be used in therapeutic contexts including type I diabetes,multiple sclerosis and liver cirrhosis, and HIV infection. Tachiban etal (Cancer Research (1995) 55: 5528-5530) have reported correlationbetween progression of astrocytomas and increased expression of Fas inthe tumour cells. De Maria and Testi (Immunology Today (1998) 19:121-125) review evidence of cells expressing Fas and its natural ligandin the proximity of lesions in multiple sclerosis, type I diabetes,liver diseases and HIV infection.

The surprising discovery of anti-Fas autoantibodies in human serumallows for modulation of binding of those antibodies to Fas to modulateFas-mediated effects, particularly apoptosis. Peptide fragments of Fas,and mimetics thereof, may be used to block antibody binding to Fas,inhibiting or increasing binding of Fas ligand to Fas. Prior to the workof the present inventors, it would not have been reasonable to expectadministration of peptide fragments of Fas (a self-antigen) to have anyutility.

As noted below in the experimental section, aspects of the presentinvention are exemplified by peptide fragments of Fas known as Fp5, withsequence GQFCHKPCPPGERKARDCTV (SEQ ID NO: 1) corresponding toGly₄₀-Val₅₉ of Fas, Fp8 with sequence QEGKEYTDKAHFSSKCRRCR (SEQ ID NO:2), Fp9 with sequence HFSSKCRRCRLCDEGHGLEV (SEQ ID NO: 3), Fp11, withsequence EINCTRTQNTKCRCKPNFFC (SEQ ID NO: 4), corresponding toGlu₁₀₀-Cys₁₁₉ of Fas, Fp12 with sequence KCRCKPNFFCNSTVCEHCDP (SEQ IDNO: 5), and Fp17 with sequence WLCLLLLPIPLIVWVKRKEV (SEQ ID NO: 6)corresponding to Trp₁₆₀-Val₁₇₉ of Fas, and Fp18 with sequenceLIVWVKRKEVQKTCRKHRKE (SEQ ID NO: 7). Fp5 is demonstrated herein to beable to induce apoptosis. Fp8 and Fp9 comprise amino acids which areimportant for binding of Fas to its natural ligand, FasL.

Fp11 and Fp17 are demonstrated herein to be able to block apoptosis.Auto-antibodies against Fp11 and Fp17 induce apoptosis, soadministration of these and related peptides may be used to blockapoptosis of Fas carrying cells. Auto-antibodies against Fp5 may havethe property of a homeostatic regulator of apoptosis by interfering withthe binding of natural Fas ligand to Fas, so administration of Fp5peptide and related peptides may be used in increase Fas ligand bindingto Fas positive cells, inducing apoptosis. Auto-antibodies against Fp8may function as a homeostatic regulator of Fas-mediated apoptosis byoccupying the Fas region which is engaged in bringing to ligand. Suchantibodies may be used as inducers or blockers or Fas-mediatedapoptosis, depending on the state of activation of the relevant cell.

In one aspect the present invention provides a peptide which is afragment of Fas, preferably human Fas, for use in a method of treatmentof the human or animal body by therapy. The treatment may be of anindividual with a complaint, disease or disorder, or may beprophylactic, as discussed further below.

Preferred peptides for use in accordance with aspects and embodiments ofthe present invention include the Fp5, Fp8, L10 Fp9, Fp11, Fp12, Fp17and Fp18 peptides for which the sequences are provided herein.

Experiments show that peptide 16, which has an over lap of 10 aminoacids with Fp17, has low or no reactivity with human sera. (Peptide 16has the sequence KEEGSRSNLGWLCLLLLPIP (SEQ ID NO: 8)). This providesindication of particular importance for the C-terminal part of Fp17.This is supported by the findings with Fp18 (see Table 1). A furtherembodiment of the present invention therefore provides a peptideincluding or consisting of the amino acid sequence QKTCRKHRKE (SEQ IDNO: 9; examples of a peptide including such sequence be ing Fp17 andFp18).

A peptide for use in the present invention may be a fragment of Fas ormay be a variant or derivative thereof, by way of addition, deletion,insertion or substitution of one or more amino acids. Such a variant orderivative thereof will generally retain ability to modulate, eitherinduce or inhibit, apoptosis and/or cellular proliferation (e.g. asmeasured using Jurkat cells or T-cells).

Preferably, the amino acid sequence of a variant or derivative peptideshares sequence similarity or identity with the relevant Fas fragmentsequence, preferably at least about 30%, or 40%, or 50%, or 60%, or 70%,or 75%, or 80%, or 85% similarity or identity, or at least about 90% or95% similarity or identity. As is well-understood, similarity allows for“conservative variation”, i.e. substitution of one hydrophobic residuesuch as isoleucine, valine, leucine or methionine for another, or thesubstitution of one polar residue for another, such as arginine forlysine, glutamic for aspartic acid, or glutamine for asparagine.Similarity may be as defined and determined by the TBLASTN program, ofAltschul et al. (1990) J. Mol. Biol. 215: 403-10, which is in standarduse in the art. Similarity or identity may be over the full-length ofthe relevant polypeptide or may more preferably be over a contiguoussequence of about 15, 20, 25, 30, 35 or 40 amino acids, compared withthe relevant wild-type amino acid sequence. Default parameters are used.

A peptide according to the present invention may be provided in a fusionwith additional amino acids. Additional amino acids may be fused at oneor both of the N-terminus and the C-terminus of the peptide. Theadditional amino acids may be an amino acid sequence that is not afragment of Fas protein, or may be an amino acid sequence that is partof that protein.

A peptide according to a further aspect of the invention may include orconsist essentially of a Fas fragment. Additional amino acids may beincluded, which amino acids may or may not be found contiguously withinFas, and the peptide may be about 10, 15, 20, 25, 30, 35 or 40 aminoacids in length. A peptide according to this aspect may be includedwithin a larger fusion protein, particularly where the peptide is fusedto a non-Fas (i.e. heterologous or foreign) sequence, such as apolypeptide or protein domain.

A peptide of the invention may be up to about 40 amino acids in length,e.g. about 10-40 amino acids in length, e.g. about 10-20.

A peptide may provided in isolated form, e.g. after its production byexpression from encoding nucleic acid. As noted further below, one ormore peptides in accordance with the present invention may be providedby peptide synthesis.

A plurality of peptides each with the amino acid sequence of a differentselected peptide may provided in isolated form, individually or in amixture. Different peptide fragments of Fas that are not naturallyjoined contiguously (or appropriate variants or derivatives thereof) maybe provided joined contiguously together in peptides or polypeptides.

A further aspect of the present invention provides the Fp17 peptide,also variants and derivatives thereof that retain the ability tomodulate apoptosis and/or cell (e.g. Jurkat or T-cell) proliferation.

Peptides and polypeptides (e.g. fusion molecules including a peptide asdiscussed) in accordance with the present invention may be made usingany of a variety of techniques at the disposal of the ordinary personskilled in the art.

Peptides may be synthesized using standard peptide chemistry such as bythe common method employing Fmoc (Fluorenilmetil-ossicarbonil)_(t)-Bu(tert-butil), as described in Atherton and Sheppard (1989), Solid PhasePeptide Synthesis, a Practical Approach, IRL Press, Oxford.

A convenient way of producing a peptide or polypeptide according to thepresent invention is to express nucleic acid encoding it, by use of thenucleic acid in an expression system. Accordingly, the present inventionalso encompasses a method of making a peptide or polypeptide (asdisclosed), the method including expression from nucleic acid encodingthe peptide or polypeptide (generally nucleic acid according to theinvention). This may conveniently be achieved by growing a host cell inculture, containing such a vector, under appropriate conditions whichcause or allow expression of the polypeptide. Peptides and polypeptidesmay also be expressed in in vitro systems, such as reticulocyte lysate.

Polynucleotides encoding peptides and polypeptides according to thepresent invention represent further aspects of the invention. In oneaspect there is provided a polynucleotide encoding a peptide asdisclosed. In a still further aspect, a polynucleotide is provided whichincludes a plurality of nucleotide sequences encoding peptides orpolypeptides according to the invention. This allows for production of amixture of peptides or polypeptides in a single expression reaction.

Nucleic acid encoding a peptide or polypeptide according to the presentinvention may be used in nucleic acid immunization in order to modulateapoptosis and/or cell proliferation in a mammal, such as a humanindividual for a therapeutic or prophylactic purpose, or a non-humanmammal for such a purpose or in order to produce antibodies forsubsequent manipulation and/or use (e.g. in diagnostic or therapeuticcontexts as discussed further below.)

Nucleic acid encoding a peptide or polypeptide according to the presentinvention may be used in a method of gene therapy, in modulation ofapoptosis and/or cellular proliferation such as in prevention and/ortreatment of a disorder in which such modulation has a beneficialeffect. This requires use of suitable regulatory elements for expressionand a suitable vector for deliver of the expression unit (codingsequence and regulatory elements) to host cells. A variety of vectors,both viral vectors and plasmid vectors, are known in the art, see e.g.U.S. Pat. No. 5,252,479 and WO 93/07282. In particular, a number ofviruses have been used as gene transfer vectors, includingpapovaviruses, such as SV40, vaccinia virus, herpes viruses, includingHSV and EBV, and retroviruses. Many gene therapy protocols in the priorart have used disabled murine retroviruses. A variety of adenovirus andadeno-associated viral vectors have been developed. Alternatives toviral vectors include transfer mediated by liposomes and direct DNAuptake and receptor-mediated DNA transfer.

Host cells containing nucleic acid encoding a peptide or polypeptide (ormixture thereof) according to the present invention may themselves beused in therapeutic or prophylactic treatment of individuals. Such hostcells are chosen in order to target the delivery of the nucleic acidencoding the peptide to the relevant site of the body in which targetlesions develop. For example, in multiple sclerosis apoptosis of Fasexpressing oligodendrocytes may be mediated by an increase in anti-Fp17auto-antibodies. Provision of Fp17 in the brain may be used to blockthese antibodies, and this may be achieved using host cells that home tothe brain, e.g. macrophages.

Nucleic acid is generally provided as DNA or RNA, though may include oneor more nucleotide analogues, and may be wholly or partially synthetic.Nucleic acid molecules and vectors according to the present inventionmay be provided in isolated and/or purified form, e.g. in substantiallypure or homogeneous form. The term “isolate” may be used to reflect allthese possibilities. Where a DNA sequence is specified, unless contextrequires otherwise the RNA equivalent, with U substituted for T where itoccurs, is encompassed.

Where it is desired to express a peptide or polypeptide from encodingnucleic acid, the nucleic acid includes appropriate regulatory controlsequences. Suitable vectors can be chosen or constructed, containingappropriate regulatory sequences, including promoter sequences,terminator fragments, polyadenylation sequences, enhancer sequences,marker genes and other sequences as appropriate. Vectors may beplasmids, viral e.g. ‘phage, or phagemid, as appropriate. For furtherdetails see, for example, Molecular Cloning: a Laboratory Manual: 2ndedition, Sambrook et al., 19891 Cold Spring Harbor Laboratory Press.Many known techniques and protocols for manipulation of nucleic acid,for example in preparation of nucleic acid constructs, a mutagenesis,sequencing, introduction of DNA into cells and gene expression, andanalysis of proteins, are described in detail in Current Protocols inMolecular Biology, Ausubel et al. eds., John Wiley & Sons, 1992.

Systems for cloning and expression of a polypeptide in a variety ofdifferent host cells are well known. Suitable host cells includebacteria, eukaryotic cells such as mammalian and yeast, and baculovirussystems. Mammalian cell lines available in the art for expression of aheterologous polypeptide include Chinese hamster ovary cells, HeLacells, baby hamster kidney cells, COS cells and many others. A common,preferred bacterial host is E. coli.

A further aspect of the present invention provides a host cellcontaining nucleic acid as disclosed herein. The nucleic acid of theinvention may be integrated into the genome (e.g. chromosome) of thehost cell. Integration may be promoted by inclusion of sequences whichpromote recombination with the genome, in accordance with standardtechniques. The nucleic acid may be on an extra-chromosomal vectorwithin the cell.

A still further aspect provides a method which includes introducing thenucleic acid into a host cell. The introduction, which may (particularlyfor in vitro introduction) be generally referred to without limitationas “transformation”, may employ any available technique. For eukaryoticcells, suitable techniques may include calcium phosphate transfection,DEAE-Dextran, electroporation, liposome-mediated transfection andtransduction using retrovirus or other virus, e.g. vaccinia or, forinsect cells, baculovirus. For bacterial cells, suitable techniques mayinclude calcium chloride transformation, electroporation andtransfection using bacteriophage. As an alternative, direct injection ofthe nucleic acid could be employed. Marker genes such as antibioticresistance or sensitivity genes may be used in identifying clonescontaining nucleic acid of interest, as is well known in the art.

The introduction may be followed by causing or allowing expression fromthe nucleic acid, e.g. by culturing host cells (which may include cellsactually transformed although more likely the cells will be descendantsof the transformed cells) under conditions for expression of the gene,so that the encoded peptide or polypeptide is produced. If the peptideor polypeptide is expressed coupled to an appropriate signal leaderpeptide it may be secreted from the cell into the culture medium.Following production by expression, a peptide or polypeptide may beisolated and/or purified from the host cell and/or culture medium, asthe case may be, and subsequently used as desired, e.g. in theformulation of a composition which may include one or more additionalcomponents, such as a pharmaceutical composition which includes one ormore pharmaceutically acceptable excipients, vehicles or carriers (e.g.see below).

A peptide or polypeptide according to the present invention may be usedas an immunogen or otherwise in obtaining binding antibodies. Antibodiesare useful in purification and other manipulation of polypeptides andpeptides, diagnostic screening and therapeutic contexts, includingpassive immunization. This is discussed further below.

Particularly useful in such contexts are the Fp17 peptide and variantsand derivatives thereof in a accordance with the present invention,including fragments of Fp17 including the C-terminal 10 amino acids, andvariants and derivatives thereof.

According to a further aspect of the present invention there is provideda method of obtaining one or more antibody molecules containing abinding site able to bind Fas, the method including bringing intocontact a population of antibody molecules and a peptide according tothe present invention, and selecting one or more antibody molecules ofthe population able to bind said peptide.

Selected antibody molecules may be of IgG or IgM isotype.

The method may involve bringing the population of antibodies intocontact with a plurality of peptides according to the invention.

The peptide or peptides may be administered to a non-human mammal tobring them into contact with a population of antibody molecules producedby the mammal's immune system, then one or more antibody molecules ableto bind the peptide or peptides may be taken from the mammal, or cellsproducing such antibody molecules may be taken from the mammal. Themammal may be sacrificed.

If cells are taken from the mammal, antibody molecules may be taken fromsaid cells or descendants thereof. Such descendants in particular mayinclude hybridoma cells.

Instead or as well as immunizing an animal, a method of obtainingantibodies as disclosed may involve displaying the population ofantibody molecules on the surface of bacteriophage particles, eachparticle containing nucleic acid encoding the antibody moleculedisplayed on its surface.

Nucleic acid may be taken from a bacteriophage particle displaying anantibody molecule able to bind a peptide or peptides of interest, formanipulation and/or use in production of the encoded antibody moleculeor a derivative thereof (e.g. a fusion protein, a molecule including aconstant region or other amino acids, and so on). Instead of usingbacteriophage for display, ribosomes or polysomes may be used, e.g. asdisclosed in U.S. Pat. No. 5,643,768, USA-5,658,754, WO95/11922.

Antibody molecules may be provided in isolated form, either individuallyor in a mixture. A plurality of antibody molecules may be provided inisolated form. Preferred antibodies according to the invention areisolated, in the sense of being free from contaminants such asantibodies able to bind other polypeptides and/or free of serumcomponents. Monoclonal antibodies are preferred for some purposes,though polyclonal antibodies are within the scope of the presentinvention.

The present invention also extends to methods of obtaining and/orraising antibodies to one or more peptides or polypeptides of theinvention. Such methods may include administering a peptide orpolypeptide or mixture of peptides or polypeptides to a mammal in orderto raise an antibody response. In a therapeutic or prophylactic contextthe mammal may be human or non-human. For the production of antibodiesor antibody-producing cells to be isolated and used for any of a varietyof purposes, a step of sacrificing a non-human mammal may be included.Such a non-human mammal may be for example mouse, rat, rabbit, dog, cat,pig, horse, donkey, goat, sheep, camel, Old World monkey, chimpanzee orother primate. Antibodies may be obtained from immunized animals usingany of a variety of techniques known in the art, and screened,preferably using binding of antibody to peptide or polypeptide ofinterest. For instance, Western blotting techniques orimmunoprecipitation may be used (Armitage et al, Nature, 357:80-82,1992).

The production of polyclonal and monoclonal antibodies is wellestablished in the art. Monoclonal antibodies can be subjected to thetechniques of recombinant DNA technology to produce other antibodies orchimeric molecules which retain the specificity of the originalantibody. Such techniques may involve introducing DNA encoding theimmunoglobulin variable region, or the complementarity determiningregions (CDRs), of an antibody to the constant regions, or constantregions plus framework regions, of a different immunoglobulin. See, forinstance, EP-A-184187, GB-A-2188638 or EP-A-239400. Humanized antibodiesin which CDRs from a non-human source are grafted onto human frameworkregions, typically with the alteration of some of the framework aminoacid residues, to provide antibodies which are less immunogenic than theparent non-human antibodies, are also included within the presentinvention. A hybridoma producing a monoclonal antibody according to thepresent invention may be subject to genetic mutation or other changes,which may or may not alter the binding specificity of antibodiesproduced. Cloning and expression of chimeric antibodies are described inEP-A-0120694 and EP-A-0125023.

As an alternative or supplement to immunising a mammal with a peptide,an antibody specific for a protein may be obtained from a recombinantlyproduced library of expressed immunoglobulin variable domains, e.g.using bacteriophage which display functional immunoglobulin bindingdomains on their surfaces—for instance see WO92/01047- orribosomes/polysomes as noted above. The library may be naive, that isconstructed from sequences obtained from an organism which has not beenimmunised with any of the proteins (or fragments), or may be oneconstructed using sequences obtained from an organism which has beenexposed to the antigen of interest.

Antibodies according to the present invention may be modified in anumber of ways. Thus the invention covers antibody fragments,derivatives, functional equivalents and homologues of antibodies,including synthetic molecules and molecules whose shape mimics that ofan antibody enabling it to bind an antigen or epitope. Example antibodyfragments, capable of binding an antigen or other binding partner arethe Fab fragment consisting of the VL, VH, Cl and CH1 domains; the Fdfragment consisting of the VH and CH1 domains; the Fv fragmentconsisting of the VL and VH domains of a single arm of an antibody; thedAb fragment which consists of a VH domain; isolated CDR regions andF(ab′)₂ fragments, a bivalent fragment including two Fab fragmentslinked by a disulphide bridge at the hinge region. Single chain Fvfragments are also included.

An antibody molecule according to the invention may be of the IgG or IgMisotype, and this may be preferred for certain embodiments.

Hybridomas capable of producing antibody with desired bindingcharacteristics are within the scope of the present invention, as arehost cells, eukaryotic or prokaryotic, containing nucleic acid encodingantibodies (including antibody fragments) and capable of theirexpression. The invention also provides methods of production of theantibodies including growing a cell capable of producing the antibodyunder conditions in which the antibody is produced, and preferablysecreted.

The reactivities of antibodies on a sample (e.g. in a diagnostic test)may be determined by any appropriate means. Tagging with individualreporter molecules is one possibility. The reporter molecules maydirectly or indirectly generate detectable, and preferably measurable,signals. The linkage of reporter molecules may be directly orindirectly, covalently, e.g. via a peptide bond or non-covalently.Linkage via a peptide bond may be as a result of recombinant expressionof a gene fusion encoding antibody and reporter molecule.

One favoured mode is by covalent linkage of each antibody with anindividual fluorochrome, phosphor or laser dye with spectrally isolatedabsorption or emission characteristics. Suitable fluorochromes includefluorescein, rhodamine, phycoerythrin and Texas Red. Suitablechromogenic dyes include diaminobenzidine. Other reporters includemacromolecular colloidal particles or particulate material such as latexbeads that are coloured, magnetic or paramagnetic, and biologically orchemically active agents that can directly or indirectly causedetectable signals to be visually observed, electronically detected orotherwise recorded. These molecules may be enzymes which catalysereactions that develop or change colours or cause changes in electricalproperties, for example. They may be molecularly excitable, such thatelectronic transitions between energy states result in characteristicspectral absorptions or emissions. They may include chemical entitiesused in conjunction with biosensors. Biotin/avidin orbiotin/streptavidin and alkaline phosphatase detection systems may beemployed.

The mode of determining binding is not a feature of the presentinvention and those skilled in the art are able to choose a suitablemode according to their preference and general knowledge.

Antibodies according to the present invention may be used in screeningfor the presence of a peptide or polypeptide, for example in a testsample containing cells or cell lysate as discussed, and may be used inpurifying and/or isolating a peptide or polypeptide according to thepresent invention, for instance following production of the polypeptideby expression from encoding nucleic acid therefor.

Antibodies are also useful in prophylaxis, by way of passiveimmunisation, and in therapy. Where antibodies are to be administered,it may be preferable to include a mixture of antibodies, such asantibodies collectively cross-reactive with a plurality of peptidesaccording to the present invention.

Antibodies which bind a peptide in accordance with the present inventionmay themselves be used as immunogens in the production of anti-idiotypicantibodies. These may be used to mimic a peptide epitope in raising animmune response in an individual, e.g. for therapeutic and/orprophylactic purposes.

An antibody may be provided in a kit, which may include instructions foruse of the antibody, e.g. in determining the presence of a particularsubstance in a test sample. One or more other reagents may be included,such as labelling molecules, buffer solutions, elutants and so on.Reagents may be provided within containers which protect them from theexternal environment, such as a sealed vial.

Antibodies against peptides (e.g. Fp17) in accordance with the presentinvention may be used in diagnostic contexts, for example to determinethe presence in an individual, or a sample removed from an individual,of cells such as tumour cells expressing high levels of Fas. A methodmay involving employing an anti-Fp17 antibody (for example), contactinga test sample with the antibody and determining binding of the antibodyto the sample.

The present invention also provides assay methods for compounds able tomodulate apoptosis and/or cell proliferation.

Further aspects of the present invention provide the use of a peptide ofthe invention as disclosed, and/or encoding nucleic acid therefor, inscreening or searching for and/or obtaining/identifying a substance,e.g. peptide or chemical compound, which interacts and/or binds with thepeptide and/or interferes with its ability to bind antibodies directedagainst it, and/or modulates its ability to affect Fas-mediatedapoptosis. Further aspects of the invention similarly provide use of anantibody against a peptide of the invention in screening for a substanceable to modulate binding of antibody to the peptide and/or binding ofantibody to Fas and/or ability of binding of antibody to Fas to induceor inhibit Fas-mediated apoptosis.

For instance, a method according to one aspect of the invention includesproviding a peptide or antibody of the invention and bringing it intocontact with a substance, which contact may result in binding betweenthe peptide or antibody and the substance. Binding may be determined byany of a number of techniques available in the art, both qualitative andquantitative.

In various aspects the present invention is concerned with provision ofassays for substances which inhibit interaction between a peptide of theinvention and an antibody directed against it.

Further assays are for substances which interact with or bind a peptideof the invention.

One aspect of the present invention provides an assay which includes:

-   (a) bringing into contact a peptide according to the invention and a    putative binding molecule or other test substance; and-   (b) determining interaction or binding between the polypeptide or    peptide and the test substance.

Another aspect of the present invention provides an assay method whichincludes:

-   (a) bringing into contact an antibody able to bind a peptide    according to the invention and a putative binding molecule or other    test substance; and-   (b) determining interaction or binding between the antibody and the    test substance.

A substance which interacts with the peptide or antibody of theinvention may be isolated and/or purified, manufactured and/or used tomodulate its activity as discussed.

A further aspect of the present invention provides an assay method whichincludes:

-   -   (a) bringing into contact a substance including a Fas fragment,        mutant, variant or derivative thereof, an antibody which is able        to bind the substance; and a test compound, under conditions in        which in the absence of the test compound being an inhibitor,        said substance and said antibody interact;

-   (b) determining interaction between said substance and said    antibody.

Such an assay method may determine interaction between complete Fas andantibody, or a Fas fragment, such as a peptide fragment selected fromFp5, Fp8, Fp9, Fp11, Fp12, Fp17, Fp18 and the C-terminal 10 amino acidsof Fp17 (N-terminal amino acids of Fp18).

Such an assay may include determination of interaction between Fas andantibody, with a peptide according to the invention also being presence,the assay determine the effect of the test substance on ability of thepeptide to modulate interaction between Fas and antibody.

The precise format of an assay of the invention may be varied by thoseof skill in the art using routine skill and knowledge. For example,interaction between a peptide and another molecule such as an antibodymay be studied in vitro by labelling one with a detectable label andbringing it into contact with the other which has been immobilised on asolid support, such as a plastic surface. Suitable detectable labelsinclude ³⁵S-methionine which may be incorporated into recombinantlyproduced peptides and polypeptides. Other labels or markers includealkaline phosphatase, peroxidase, avidin-biotin, which may be coupleddirectly to an anti-peptide antibody. A further option for those skilledin the art is to use a labelled anti—anti-peptide antibody which may bereacted to peptide-anti-peptide. Activity of alkaline phosphatase,peroxidase or avidin-biotin, or other label, may be measured in aspectrophotometer.

Further assays according to aspects of the present invention involvedetermination of the ability of a test substance to modulateFas-mediated apoptosis of cells.

The binding of antibody to Fas, e.g. present on a cell membrane, may beinhibited by adding soluble Fas or a fragment thereof or other peptideaccording to the present invention. A T-cell line, such as Jurkat cells,expressing Fas on the surface may be employed. Thus, ability of a testsubstance to modulate apoptosis induced by an antibody directed againsta peptide of the invention may be determined.

The amount of apoptosis may be determined, for instance by detecting DNAfragmentation or changes in phosphatidylserine translocation occurringat the cell membrane. DNA fragmentation may be measured usingconventional agarose gel electrophoresis. Changes in phosphatidylserinetranslocation may be analysed by staining with labelled annexin Vfollowed by FACS analysis.

A method of screening for a substance which modulates Fas-mediatedapoptosis may include contacting one or more test substances withT-cells or other Fas positive cells and anti-Fas antibodies,particularly antibodies directed against a peptide in accordance withthe invention, in a suitable reaction medium, determining the level ofapoptosis and comparing that level with the level in a comparablereaction medium untreated with the test substance or substances. Adifference in apoptosis between the treated and untreated reaction mediais indicative of a modulating effect of the relevant test substance orsubstances.

Combinatorial library technology (Schultz, J S (1996) Biotechnol. Prog.12:729-743) provides an efficient way of testing a potentially vastnumber of different substances for ability to modulate Fas activity.Prior to or as well as being screened for modulation of activity, testsubstances may be screened for ability to interact with a peptide orantibody of the invention, e.g. in a yeast two-hybrid system. This maybe used as a coarse screen prior to testing a substance for actualability to modulate Fas activity.

The amount of test substance or compound which may be added to an assayof the invention will normally be determined by trial and errordepending upon the type of compound used.

Typically, from about 0.01 to 100 nM concentrations of putativeinhibitor compound may be used, for example from 0.1 to 10 nM. Greaterconcentrations may be used when a peptide is the test substance.

Compounds which may be used may be natural or synthetic chemicalcompounds used in drug screening programmes. Extracts of plants whichcontain several characterised or uncharacterised components may also beused. Other candidate inhibitor compounds may be based on modelling the3-dimensional structure of a polypeptide or peptide fragment and usingrational drug design to provide potential inhibitor compounds withparticular molecular shape, size and charge characteristics.

Following identification of a substance which modulates or affects Fasactivity, the substance may be investigated further. Furthermore, it maybe manufactured and/or used in preparation, i.e. manufacture orformulation, of a composition such as a medicament, pharmaceuticalcomposition or drug. These may be administered to individuals.

Thus, the present invention extends in various aspects not only to asubstance identified as a modulator of FAs activity, in accordance withwhat is disclosed herein, but also a pharmaceutical composition,medicament, drug or other composition comprising such a substance, amethod comprising administration of such a composition to a patient,e.g. for treatment (which may include preventative treatment) ofdisease, use of such a substance in manufacture of a composition foradministration, e.g. for treatment of disease, and a method of making apharmaceutical composition comprising admixing such a substance with apharmaceutically acceptable excipient, vehicle or carrier, andoptionally other ingredients.

Also encompassed within the scope of the present invention arefunctional mimetics of peptide fragments of Fas which are able tomodulate apoptosis and/or cellular proliferation. A “functional mimetic”is a substance which may not contain an active portion of the relevantamino acid sequence, and probably is not a peptide at all, but whichretains the relevant activity.

Non-peptide “small molecules” are often preferred for many in vivopharmaceutical uses. Accordingly, a mimetic or mimic of the substance(particularly if a peptide) may be designed for pharmaceutical use. Thedesigning of mimetics to a known pharmaceutically active compound is aknown approach to the development of pharmaceuticals based on a “lead”compound.

This might be desirable where the active compound is difficult orexpensive to synthesize or where it is unsuitable for a particularmethod of administration, e.g. peptides may not be well suited as activeagents for oral compositions as they may be quickly degraded byproteases in the alimentary canal. Mimetic design, synthesis and testingmay be used to avoid randomly screening large number of molecules for atarget property.

There are several steps commonly taken in the design of a mimetic from acompound having a given target property.

Firstly, the particular parts of the compound that are critical and/orimportant in determining the target property are determined. In the caseof a peptide, this can be done by systematically varying the amino acidresidues in the peptide, e.g. by substituting each residue in turn.These parts or residues constituting the active region of the compoundare known as its “pharmacophore”.

Once the pharmacophore has been found, its structure is modeled toaccording its physical properties, e.g. stereochemistry, bonding, sizeand/or charge, using data from a range of sources, e.g. spectroscopictechniques, X-ray diffraction data and NMR. Computational analysis,similarity mapping (which models the charge and/or volume of apharmacophore, rather than the bonding between atoms) and othertechniques can be used in this modeling process.

In a variant of this approach, the three-dimensional structure of theligand and its binding partner are modeled. This can be especiallyuseful where the ligand and/or binding partner change conformation onbinding, allowing the model to take account of this the design of themimetic.

A template molecule is then selected onto which chemical groups whichmimic the pharmacophore can be grafted. The template molecule and thechemical groups grafted on to it can conveniently be selected so thatthe mimetic is easy to synthesize, is likely to be pharmacologicallyacceptable, and does not degrade in vivo, while retaining the biologicalactivity of the lead compound. The mimetic or mimetics found by thisapproach can then be screened to see whether they have the targetproperty, or to what extent they exhibit it. Further optimization ormodification can then be carried out to arrive at one or more finalmimetics for in vivo or clinical testing.

Further aspects of the present invention therefore relate to provisionof non-peptidyl mimetics of peptides for use in the present invention.One aspect of the invention provides the use of a peptide as disclosedin the identification or design of a non-peptidyl mimetic which retainsability to modulate apoptosis and/or cell proliferation. A furtheraspect provides a method of testing a non-peptidyl mimetic of a peptidefor use in the present invention for ability to modulate apoptosisand/or cellular proliferation.

As noted already, peptides, mimetics, polypeptides, antibodies andnucleic acid in accordance with the present invention may be formulatedinto compositions, and are useful in pharmaceutical contexts. Thesecompositions may include, in addition to one of the above substances, apharmaceutically acceptable excipient, carrier, buffer, stabiliser orother materials well known to those skilled in the art. Such materialsshould be non-toxic and should not interfere with the efficacy of theactive ingredient. The precise nature of the carrier or other materialmay depend on the route of administration, e.g. oral, intravenous,cutaneous or subcutaneous, nasal, intramuscular, intraperitoneal routes.

Compositions for oral administration may be in tablet, capsule, powderor liquid form. A tablet may include a solid carrier such as gelatin oran adjuvant. Liquid pharmaceutical compositions generally include aliquid carrier such as water, petroleum, animal or vegetable oils,mineral oil or synthetic oil. Physiological saline solution, dextrose orother saccharide solution or glycols such as ethylene glycol, propyleneglycol or polyethylene glycol may be included.

For intravenous, cutaneous or subcutaneous injection, the activeingredient will be in the form of a parenterally acceptable aqueoussolution which is pyrogen-free and has suitable pH, isotonicity andstability. Those of relevant skill in the art are well able to preparesuitable solutions using, for example, isotonic vehicles such as SodiumChloride Injection, Ringer's Injection, Lactated Ringer's Injection.Preservatives, stabilisers, buffers, antioxidants and/or other additivesmay be included, as required.

A peptide may be linked to an appropriate carrier. Various methods ofcoupling peptides to other molecules are known in the art, includingdisulphide forming reagents (where the peptide includes a cysteine—or acysteine is added to the peptide for this purpose), thio-ether formingcoupling agents and so on. Carriers include human serum albumin (HSA),tetanus toxoid, other rather large proteins that have reasonablehalf-lives under physiological conditions, and stable non-proteinaceousmolecules such as polysaccharides and copolymers of amino acids.

An adjuvant may be included, such as alum, oil-in-water emulsions orFreund's Adjuvant (Complete or Incomplete). Cytokines may be used topotentiate immunogenicity of the peptide or polypeptide composition.

Whether it is a polypeptide, antibody, peptide, nucleic acid molecule,mimetic, small molecule or other pharmaceutically useful compoundaccording to the present invention that is to be given to an individual,administration may be in a “prophylactically effective amount” or a“therapeutically effective amount” (as the case may be, althoughprophylaxis may be considered therapy). Most preferably the effect issufficient to prevent the individual from suffering one or more clinicalsymptoms, and/or reduce pain. A therapeutic effect is sufficient topotentiate the immune response of an individual to pre-existingdisorder, preferably sufficient to antagonise the disorder, wholly orpartially. Most preferably the effect is sufficient to ameliorate one ormore clinical symptoms, and/or cure the disorder and/or reduce pain inthe individual. The actual amount administered, and rate and time-courseof administration, will depend on the nature and severity of what isbeing treated. Prescription of treatment, e.g. decisions on dosage etc,is within the responsibility of general practitioners and other medicaldoctors, and typically takes account of the disorder to be treated, thecondition of the individual patient, the site of delivery, the method ofadministration and other factors known to practitioners. Examples of thetechniques and protocols mentioned above can be found in Remington'sPharmaceutical Sciences, 16th edition, Osol, A. (ed), 1980.

Further aspects of the invention provide methods of treatment includingadministration of a peptide, mixture of peptides, antibody molecule ormixture of antibody molecules, as provided, pharmaceutical compositionsincluding such a peptide, mixture of peptides, antibody molecule ormixture of antibody molecules, and use of such a peptide, mixture ofpeptides, antibody molecule or mixture of antibody molecules, in themanufacture of a medicament for administration, for example in a methodof making a medicament or pharmaceutical composition includingformulating the specific binding member with a pharmaceuticallyacceptable excipient. Nucleic acid encoding peptides or polypeptides,and non-peptide mimetics may be employed.

A composition may be administered alone or in combination with othertreatments, either simultaneously or sequentially dependent upon thecondition to be treated and the availability of alternative oradditional treatments. In some embodiments of the present invention therelevant molecule is used in conjunction with anti-CD3 antibodies orother anti-CD3 binding molecules.

One aspect of the present invention provides use of a peptide asdisclosed in the manufacture of a medicament for use in a method oftreatment of the human or animal body by therapy. Treatment may be of aproliferative disorder, such as a tumour, cancer or psoriasis, or anautoimmune disorder, type diabetes, multiple sclerosis, liver cirrhosisand so on. Apoptosis may be induced or inhibited and/or cellularproliferation inhibited or stimulated.

Another aspect provides a method of treating a mammal against such adisorder, the method including administering a peptide or mixture ofpeptides, mimetic or mimetics, antibody or antibodies or nucleic acid asdisclosed, to the mammal.

A peptide, antibody or other therapeutic molecule according to thepresent invention may be targeted to a lesion, e.g. tumour. Antibodiesdiffuse rather well through tissues and injection at a site distal tothat of the lesion may be employed. Alternatively, direct injection intoa lesion, e.g. tumour, may be employed, and this may be utilised forpeptides and other molecules. Targeted viral vectors may be used todeliver nucleic acid encoding a peptide, polypeptide or antibodyaccording to the invention to a site for expression of the encodedproduct.

Further aspects and embodiments of the present invention will beapparent to those skilled in the art based on the present disclosure.Embodiments of and experimental basis for the present invention will nowbe described in more detail with reference to the following figures:

FIG. 1 illustrates results of experiments demonstrating that sera fromhealthy blood donors contain antibodies against human Fas peptides.Serum specimens from 30 individuals were analyzed. ELISA titers ofantibodies against the reactive Fas peptides Fp5, Fp11 and Fp17 areexpressed as percentiles and median values by use of the box (notched)and whisker plot of the StatView+Graphics statistical computer program.

FIG. 2(A) shows results of TUNEL assay of apoptosis of Jurkat leukemia Tcells in absence (filled) and in presence (open) of Fas-Fc chimericprotein demonstrating that anti-Fas peptide auto-antibodies can induceFas-mediated apoptosis.

FIG. 2(B) shows that anti-Fas peptide auto-antibodies can inhibitapoptosis induced by CH-11 murine anti-Fas antibodies.

FIG. 3(A) shows proliferation of T-cell blasts in response tostimulation with anti-Fas peptide autoantibodies in absence (filled) orpresence (open) of immobilized anti-CD3 mob. The gray bar represents theresult of the anti-CD3 mAb alone.

FIG. 3(B) shows FasL expression on the surface of PHA-activated T-cellblasts in response to stimulation with anti-10 Fas peptideautoantibodies in absence (filled) or presence (open) of immobilizedanti-CD3 mAb. The gray bar represents the result of the anti-CD3 mAbalone.

FIG. 4(A) shows proliferation of non-stimulated PBMC in response tostimulation with anti-Fas peptide autoantibodies in absence (filled) orpresence (open) of anti-CD3 mAb.

FIG. 4(B) shows apoptosis of non-stimulated PBMC in response tostimulation with anti-Fas peptide autoantibodies in absence (filled) orpresence (open) of anti-CD3 mAb.

EXPERIMENTAL

Synthesis of Fas peptides.

The derived amino acid sequence (a.a.1-179) of the Fas protein (Itoh etal., 1991) was used for simultaneous multiple solid-phase peptidesynthesis (Houghten et al, 1985) of 20 amino acid long peptides (with a10-residue overlap).

The peptides demonstrated 70-97% homogeneity, as revealed by HPLC. Thesepeptides were used in ELISA of 30 human sera obtained from healthy blooddonors and the specificity of the assay was confirmed by peptidecompetition ELISA as described (Leonov et al., 1994). Peptides Fp5(Gly₄₀-Val₅₉), Fp11 (Glu₁₀₀-Cys₁₁₉), and Fp17 (Trp₁₆₀-Val₁₇₉) wereselected as positively reacting with human sera and were used insubsequent experiments.

Purification and Characterization of Fas Autoantibodies.

Affinity columns were made by coupling 7 mg each of Fp5, Fp11, and Fp17to ECH-Sepharose (Pharmacia Biotech, Sweden) withN-ethyl-N′-(3-dimethyaminopropyl)carbodiimide hydrochloride, asdescribed in the manufacturer's affinity chromatography protocol. Afterovernight absorption of pooled human sera of healthy blood donors, theanti-peptide antibodies were eluted by 4 M KSCN, concentrated, dialyzedand sterilized by filtration through 0.22 μm pore size filter. Theseeluates were analyzed by electrophoresis and Western Blot using NOVEXNUPAGE Gel kit: 10% Bis-Tris Gels/MOPS SDS Buffers according tomanufacturer's protocols (NOVEX, San Diego, Calif.). Purified human IgGand mouse IgM, and NOVEX Mark 121 Wide Range Protein Standard were usedas reference.

Solubilized proteins from 10 Jurkat leukemia T cells were separated andtransferred to 0.45 μm pore size supported nitrocellulose membrane(Bio-Rad Laboratories AB, Sweden) using NOVEX Western Blot buffersystem. Membranes were probed by IgM class anti-Fas mAb CH-ll (MedicalBiological Laboratory, Nagoya, Japan) and IgG1 class anti-Fas mAb clone13 (Transduction Laboratories, Lexington, Ky.) according tomanufacturer's recommendations; human control IgG (not reacting with Faspeptides), eluates from affinity columns, pooled human sera before andafter column immunoadsorption diluted by 1/50 in PBS containing 5%non-fat dry milk, 0.1% Tween-20 and 0.001% anti-foam agent (Sigma).

Bound antibodies were revealed by sequential use of F(ab)₂ fragments ofgoat anti-human light chain antibodies conjugated with peroxidase andSuperSignal ULTRA chemiluminescent substrate system (both are fromPierce, Rockford, Ill.).

Detection of Apoptosis

Jurkat leukemia T cells (2×10⁶/well) in RPMI 1640 supplemented with 10%FCS, 10 mM-HEPES, 2 mM-glutamin, 50 FM-2-mercaptoethanol, 0.1M-non-essential amino acids, and 10 μg/ml gentamicin (RPMI-HEPES) werecultured in 24-well plates previously coated overnight with anti-Faspeptide antibodies isolated by affinity-column. Theantibody-concentrations were anti-Fp5 (10 μg/ml), anti-Fp11 (10 μg/ml)and anti-Fpl7 (40 μg/ml). Cells were collected after 48 h incubation,and the fragmented DNA of apoptotic cells was assessed by ApoptosisDetection System, Fluorescein (Promega, Madison, Wis.) based onTdT-mediated dUTP Nick-End Labeling (TUNEL) assay and FACS analysis(CellQuest software, Becton Dickinson) according to manufacturer'sinstructions. For apoptosis inhibition experiments, Jurkat cell culturemedium was supplemented with 200 ng/ml of recombinant Fas-Fc chimera(R&D Systems Europe Ltd, Abingdon, UK). Inhibition of CH-11-mediatedapoptosis was performed as described in Fadeel et al., 1997. Data arepresented as the mean value of four independent experiments, and thestandard error of mean is indicated by the error bars.

Proliferation Assay.

PBMC were stimulated with phytohemagglutinin (PHA) (10/g/ml) (PharmaciaBiotech, Sweden) for 48 hours. Approximately 5×10⁵ stimulated cells werethen cultured in a flat-bottom 96-well plate previously coated overnightwith anti-Fas peptide affinity isolated antibodies and purified anti-CD3(OKT-3, 10 μg/ml) in RPMI-HEPES medium. After 48 hours incubation, theproliferation was assessed by Alamar Blue Assay (BiosourceInternational, USA.) and the results were expressed as percentdifference in reduction of (fluorometric/colorometric) REDOX indicatorbetween antibody coated and control (without antibodies) wells accordingto manufacturer's instructions (AMS Biotechnology AB, Sweden).

The FasL expression in PHA-stimulated T-cell blasts was detected at thesame time point by use of IgG1 anti-FasL mAb, clone 33 (TransductionLaboratories, Lexington, Ky.) and Phycoerythrine (PE) conjugated F(ab)₂fragments of goat anti-mouse IgG (Dako, Sweden). Results were expressedas percent of positive cells analyzed by FACS.

Non-Stimulated PBMC were Isolated by Ficoll-Hypaque

(Pharmacia Biotech, Sweden) and used for proliferation assays asdescribed above for PHA stimulated cells. Apoptosis was quantified byTUNEL assay and expressed as percent difference in fluorescence betweenantibody containing and antibody-free cultures.

Computer-Modelling of Fas

The molecular model for the extracellular domain (Itoh et al., 1991) ofthe Fas monomer (amino acids His₃₈-Lys₁₄₉) was created usingknowledge-based protein modeling methods as implemented in theSwiss-Model server (Peitsch et al., 1995 and 1996). The model was basedon the three-dimensional structure of the TNFR1 (entry No. 1TNR inBrookhaven Protein Data Bank).

Results

Anti-Fas Antibodies of IgG Class are Present in the Serum of BloodDonors.

Peptides corresponding to the extracellular and transmembrane parts ofhuman Fas (Itoh, et al., 1991) were synthesized. Three of thesepeptides, Fp5 (Gly₄₀-Val₅₉ with sequence GQFCHKPCPPGERKARDCTV, SEQ IDNO: 1), Fp11 (Glu₁₀₀-Cys₁₁₉ with sequence EINCTRTQNTKCRCKPNFFC. SEQ IDNO: 4) and Fp17 (Trp₁₆₀-Val₁₇₉ with sequence WLCLLLLPIPLIVWVKRXEV,SEQUENCE ID NO: 6), were reactive with antibodies present in the bloodof the 30 healthy donors (FIG. 1). The serum titers against the threedifferent peptides were variable with the lowest mean titers detectedagainst Fp17.

These antibodies were purified by affinity chromatography based onimmobilized Fas peptides. Polyacrylamide gel electrophoresis (PAGE)analysis of the eluates demonstrated that the majority of purifiedproteins had a molecular weight corresponding to IgG class antibodies.The specificity of the isolated antibodies was further assayed byWestern blot with whole Fas protein which confirmed that anti-Fasantibodies are present in the serum of healthy human subjects.

Similar experiments were performed with commercially availableimmunoglobulin preparations derived from pooled human serum enriched byimmunosorbence for immunoglobulin fractions (Gammagard-Baxter, Hyland,Glendale, Calif. USA).

Results for the experiments with human serum and with the immunoglobulinpreparations are shown in Table 1.

The presence of both IgG and IgM auto-antibodies was established.

Autoantibodies to Fas regions represented by Fp11 and Fp17 mediateapoptosis of Jurkat leukemia cells through Pas.

Ability to mediate apoptosis and inhibition of proliferation wasdetermined using antibodies immobilized to plastic surface. Immobilizedhuman antibodies directed to Fp11 and Fp17 induced apoptosis inrespectively 49% and 58% of Jurkat leukemia cells (FIG. 2(A)). The levelof apoptosis induced by Fp5 did not differ significantly from controlwell in which antibodies were not included, 25% versus 18%.

Next, ability of anti-Fas autoantibodies to affect cell proliferation inthe Jurkat cell system was analysed. Human antibodies directed to Fp17inhibited cell proliferation by 40% as compared to control well; thelevels of inhibition of proliferation were lower for anti-Fp5 andanti-Fp11, 8% and 10% respectively. The use of either protein A oranti-human Ig immunosorbent to immobilize the anti-peptide antibodiesgave similar results. Thus, IgG class anti-Fas peptide antibodiespresent in human blood can induce apoptosis and inhibit proliferation inJurkat T cells. Without being bound by any particular theory, thisbiological effect is likely to be mediated through cross-linking of Fasmolecules on the cell surface, a mechanism suggested to operate foranti-Fas monoclonal antibodies of both IgM and IgG subclasses, includingIgG1 (Dhein et al., 1992; Fadeel et al., 1997).

Two sets of blocking experiments were performed to establish thatapoptosis induced by anti-Fas autoantibodies is mediated through Fas.

The chimeric protein Fas-Fc, consisting of the extracellular part of Fas(aa 1-173) and Fc-part of human IgG was previously shown to blockapoptosis caused by Fas-FasL interaction (Itoh et al., 1991). Additionof this protein to Jurkat cells completely reduced apoptosis induced byhuman antibodies to Fp5 and Fp11 (FIG. 2(A)). For Jurkat cells incubatedwith anti-Fp17, the reduction of apoptosis was 60%, although it can benoted that the portion of Fas included in the Fas-Fc fragment is 1-173whereas Fp₁₁₇ span is between Trp₁₆₀ snd Val₁₇₉.

In the second set of experiments purified anti-Pas autoantibodies insoluble form were used to block apoptosis induced by the anti-Fas IgMmonoclonal CH-11. Anti-Fas IgG1 murine monoclonal antibodies werepreviously demonstrated to inhibit the effect of CH-11 (Fadeel et al.,1997). The autoantibodies directed to Fp-5 and Fp-11 reducedCH-11-mediated apoptosis by more than 50%, thus to levels similar to thespontaneous apoptosis occurring in the non-treated cells. Humanauto-antibodies to Fp-17 only diminished the CH-11 induced apoptosis by31% (FIG. 2(B)). It has previously been shown that Fp11 can block theapoptotic activity of CH-11 (Fadeel et al., 1995). Thus, the reductionof apoptosis noticed upon incubation with anti-Fp11 is probably due to aspecific blocking of the Fas site recognized by CH-ll. Antibodiesbinding to Fp5 and Fp17 of Fas, on the other hand, may sterically hinderbinding of CH-11.

Together, these data demonstrate that apoptosis of immortalized Jurkatleukemia T cells induced by human anti-Fas autoantibodies is mediatedthrough a Fas-dependent pathway.

Co-stimulation with Anti-CD3 Antibodies and Anti-Fas AutoantibodiesAffects Proliferation According to the State of Cell-Activation.

Next, ability of human anti-Fas peptide autoantibodies to affectproliferation of primary T cells was investigated.

Cross-linking of CD3/TCR receptor is known to trigger a Fas-dependentprocess termed activation-induced cell death (AICD) and reduced cellproliferation (Varadhachary et al., 1997; Dhein et al., 1995; Brunner,et al., 1997; Ju et al., 1997).

PHA-stimulated T cell blasts were incubated with the isolated anti-Fasantibodies together with antibodies directed to CD3. The auto-antibodieshad no effect when immobilized alone (FIG. 3(A)). However, costimulationof cells with immobilized anti-CD3 antibodies and anti-Fasautoantibodies significantly reduced proliferation (21-42% reduction) incomparison with anti-CD3 alone (12% reduction). Anti-Fas autoantibodiescan therefore enhance CD3-mediated anti-proliferative effect.

Interestingly, it was found that reduction of proliferation wasparalleled with increased expression of FasL (FIG. 3(B)). The datasuggest that costimulation of the CD3/TCR complex and Fas by anti-CD3and human anti-Fas autoantibodies might decrease the proliferation ofactivated T cell blasts. A possible mediator of this effect may be FasL,as demonstrated by up-regulation of this molecule upon cross-linking ofFas and CD3.

CD3/TCR and Fas engagement in cellular proliferation and apoptosis wasfurther investigated in experiments with non-stimulated peripheral bloodmononuclear cells (PBMC). In this system immobilized human anti-Fasauto-antibodies alone or in combination with anti-CD3 increasedproliferation between 20 and 40% (FIG. 4(A)). This effect may be due toreduction of spontaneous apoptosis. Indeed, the costimulation withanti-CD3 antibody and human anti-Fas auto-antibodies induced reductionof spontaneous apoptosis by 15 to 55% for the three anti-peptideantibodies (FIG. 4(B)). These data correlate with previous observationsthat CD3 and Fas costimulation using murine monoclonal antibodiesinduced increase of proliferation of isolated T cells (Alderson et al.,1993; Alderson et al., 1994).

Thus, human anti-Fas auto-antibodies can modulate the biological effectof CD3/TCR ligation in non-stimulated PBMC through a mechanism includingreduction of apoptosis.

Pas-binding Sites for Autoantibodies and Fas L.

To gain knowledge on how anti-Fas autoantibodies may induceapoptotic/activation signals through Fas, a molecular model of thisprotein was created where the antibody-binding sites could be visualizedin relation to the surfaces of FasL interaction. The extracellular partof the Fas molecule has distinct sequence homology to TNF receptor 1(TNFR1; Nagata and Golstein, 1995). Based on the three-dimensionalstructure of TNFR1 a molecular model (Peitsch, 1995 and 1996) of Fas wascreated.

The model shows that antibody binding to the Fas regions represented byFp5 (Gly₄₀-Val₅₉) and Fp11 (Glu₁₀₀-Cys₁₁₉) may sterically interfere withFasL binding to Arg₈₆ and Arg₈₇, amino acids that are critical forFas-FasL binding (Starling, et al., 1997). This may also be relevant foramino acids Lys₈₄, Leu₉₀, Glu₉₃ and His₁₂₆, that are thought to beimportant for Pas-FasL interactions (Starling, et al., 1997). Thus, thelocalization of these two antibody-binding regions of the Fas moleculemay explain the biological effect of the anti-Fas autoantibodies oncells by either interference with FasL binding or by direct induction ofapoptotic or proliferative signals. In this context it is important tonote that the apoptotic activity of the prototypic anti-human Fas mAb(clone CH-11) depends on binding to the region represented by Fp11, thusindicating that this segment display pronounced antigenic property(Fadeel et al., 1995). Of course performance of aspects of the presentinvention does not require knowledge of how the invention works and thescope of the invention is not limited by any particular theory.

The region of Fas represented by Fp17 (Trp₁₆₀-Val₁₁₉) is predicted to bea transmembrane domain and could not be included in the model. Therecognition of this Fas domain by auto-antibodies mediating biologicaleffect indicates that part of this domain is likely to be exposed on thecell surface. The titers against Fp17 were the lowest among the threepeptides. However, the apoptosis-inducing effect of these anti Fp17antibodies was evident and exceeded that of anti-Fp5 and anti Fp11 whenused at the same concentration.

Fp17 does not contain residues involved in the contact with FasL. Theresults provide indication that autoantibody binding to this Fp17 regionmay efficiently transduce a signal to the intracellular cell deathmachinery.

Discussion

The experimental work described above demonstrates the existence ofbiologically active human autoantibodies against Fas, one of the keymolecules in the control of lymphocyte expansion/deletion in vivo (Lynchet al., 1995). The anti-Fas autoantibodies were present in the serum ofa majority of healthy individuals.

The work also demonstrates that human anti-Fas autoantibodies cantrigger cellular responses through the Fas receptor in vitro. Thisprovides indication that in addition to already described modes ofregulation of Fas-mediated signals (Suda et al., 1993; Irmler et al.,1997) autoantibodies constitute an additional level of modulation ofFas-mediated functions in vivo.

Paralleling previous findings on murine monoclonal antibodies(Owen-Schaub et al., 1992; Klas et al., 1993; Alderson et al., 1993 and1994), the human autoantibodies against Fas may be used to provide bothapoptosis inducing and stimulatory effects depending on the activationstate of the cells. Conversion of Fas-mediated signals from cellactivation to apoptosis may represent a safety mechanism by which theimmune system eliminates transformed cells from the host (Alderson etal., 1994). Considering that antibodies can easily diffuse to tissues,hypothetical roles for the anti-Fas autoantibodies may be found in thisbalance of cell activation and apoptosis, in the termination of ongoingimmune responses through AICD (Kabelitz et al., 1993) and in themaintenance of peripheral tolerance (Fisher et al., 1995; Rieux-Laucatet al., 1995).

The activity of anti-Fas autoantibodies has potential implications indiseases which are linked to Fas dysregulation, e.g. liver damage (Galleet al., 1995), insulin-dependent diabetes mellitus (Stassi et al., 1997)and multiple sclerosis (Dowling et al., 1996; D'Souza et al., 1996).Increased levels of Fas and FasL in these diseases may be paralleled bychanges in the levels of Fas autoantibodies.

Autoantibodies to the three regions of Fas display different biologicalactivity. Anti Fp17 autoantibodies are efficient in mediating apoptosisof Jurkat cells whereas anti Fp5 antibodies were found to be poorinducers of apoptosis but can efficiently block the apoptotic activityof anti-fas mAbCH-11. Thus the binding of autoantibodies to regions ofFas which may interfere spacially with the ligand (Fp5) or directlytransduce apoptotic signals (Fp17) may be exploited in therapeuticalsettings aimed at inducing or reducing Fas-mediated apoptosis.

Measurement of distribution and titres of antibodies directed to Faspeptides in accordance with the present invention provides furtherinsight into Fas dysregulation and involvement in pathogenesis ofdiseases. Such distribution and titres are measured in sera obtainedfrom patients with multiple sclerosis, type I diabetes and HIVinfection, using techniques available to those skilled in the art.

Fragments and variants of peptides of the invention which retain abilityto inhibit or induce apoptosis are identified, along with minimalepitopes for binding of anti-Fas antibodies, using systematicsubstitution in the peptides of each individual amino acid, for instanceusing alanine scanning. Reactivity of human sera is evaluated withenzyme linked immunosorbent assay to define amino acids important ornecessary for binding.

Colon carcinoma, hepatocellular and other carcinomas in which theexpression of Fas is down-regulated (Walker et al. (1997) J. Immunology158: 4521-4524) are treated with peptides of the invention, particularlyFp17, the C-terminal amino acids of Fp17, and fragments and variantsthereof. The portion of Fas including Fp17 includes the membranespanning peptide and may be used to provide down-stream signalling tocells to undergo apoptosis.

Antibodies against Fp17 are injected into animals to induce apoptosis ofanimal tumours.

Animal models representative of (i) multiple sclerosis and (ii) type Idiabetes are treated with a peptide or antibody of the invention inorder to block anti-Fas auto-antibody triggering of apoptosis indiseased organs.

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All documents mentioned anywhere herein are incorporated by reference.

TABLE 1 Reactivity of human serum and immunoglobulin preparations to Faspeptides in ELISA. Peptide Immunoglobulin number Human serumpreparations (Gammagard) Fp1 − − Fp2 − − Fp3 − − Fp4 − − Fp5 ++ + Fp6 −− Fp7 − − Fp8 − +++ Fp9 − ++ Fp10 − − Fp11 +++ +++ Fp12 ++ ++ Fp13 − −Fp14 − − Fp15 − − Fp16 − + Fp17 +++ + Fp18 not done +++ + = <0.5 O.D.490 nm; ++ = <1.0 O.D.; +++ > 1.0 O.D.

1. A method of obtaining one or more human antibody molecules containinga binding site that binds human Fas, the method comprising bringing intocontact a population of human antibody molecules and a peptide of 10-20amino acids in length which is a fragment of human Fas, said fragmentcomprising an amino acid sequence selected from the group consisting of:(i) GQFCHKPCPPGERKARDCTV (SEQ ID NO. 1), (ii) QEGKEYTDKAHFSSKCRRCR (SEQID NO. 2), (iii) HFSSKCRRCRLCDEGHGLEV (SEQ ID NO. 3), (iv)EINCTRTQNTKCRCKPNFFC (SEQ ID NO. 4), (v) KCRCKPNFFCNSTVCEHCDP (SEQ IDNO. 5), (vi) WLCLLLLPIPLIVWVKRKEV (SEQ ID NO. 6), (vii)LIVWVKRKEVQKTCRKHRKE (SEQ ID NO. 7), and (viii) QKTCRKHRKE (SEQ ID NO.9); said fragment comprising an immunogenic amino acid sequence which isfound within an amino acid sequence selected from said group; andselecting one or more human antibody molecules able to bind saidpeptide.
 2. A method of according to claim 1 wherein an antibodymolecule directed to said peptide, or a mixture of antibody moleculesdirected to one or more said peptides, is obtained and is formulatedinto a composition comprising pharmaceutically acceptable excipient,carrier, buffer or stabiliser.
 3. A method according to claim 1 furthercomprising providing host cells in vitro that produce the selected humanantibody molecules able to bind said peptide.
 4. A method according toclaim 3 wherein an antibody molecule directed to said peptide, or amixture of antibody molecules directed at least one of said peptides, isobtained and is formulated into a composition comprising apharmaceutically acceptable excipient, carrier, buffer or stabiliser.